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J Biol Chem, Vol. 274, Issue 16, 10777-10783, April 16, 1999

The Conserved Lysine 860 in the Additional Fatty-acylation Site of Bordetella pertussis Adenylate Cyclase Is Crucial for Toxin Function Independently of Its Acylation Status

Tümay BasarDagger , Vladimír HavlícekDagger , Silvia BezouskováDagger , Petr HaladaDagger , Murray Hackett§, and Peter SeboDagger

From the Dagger  Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20 Prague 4, Czech Republic and the § Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon -amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta 9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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